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. 2020 May 5;136(8):957–973. doi: 10.1182/blood.2019002548

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Figure 4. — Snai1 overexpression induces self-renewal potential in immature myeloid cells and impairs granulocytic differentiation. (A) Snai1^tg/+ and Snail^tg/tg bone marrow generate significantly more colony-forming unit (CFU) granulocyte/macrophage (GM) colonies and significantly fewer CFU granulocyte/erythroid/macrophage/megakaryocyte (GEMM) colonies as compared with Snai1^+/+ bone marrow. (B) Wright-Giemsa staining analysis of Snai1^tg/+ and Snail^tg/tg methylcellulose colony cytospins showing almost complete lack of mature granulocytes (green arrows) and an increase in immature myeloid cells (red arrows) as compared with Snai1^+/+ colonies. (C) The number of cells per colony (calculated as total number of cells per total number of colonies) was significantly increased in Snail^tg/tg cultures compared with Snai1^+/+ cultures. Snai1^tg/+ cultures also showed a trend toward an increased number of cells per colony; however, this was not significant. (D) Quantification of bone marrow methylcellulose colonies showing that Snai1^tg/+ hematopoietic progenitor cells have increased self-renewal capability compared with Snai1^+/+ controls, with Snai1^tg/+ and Snai1^tg/tg cells able to generate colonies in methylcellulose up to 5 rounds of replating. Snai1^+/+ cells were only able to replate up to 3 rounds. (E) Flow cytometric analysis of methylcellulose-derived hematopoietic cells showing a higher percentage of immature and mature myeloid cells generated from Snai1^tg/+ and Snai1^tg/tg progenitor cells at the first round of culture. (F) Quantification of immature (CD11b⁺GR1^lo) and mature (CD11b⁺GR1^hi) myeloid cell populations in Snai1^+/+, Snai1^tg/+, and Snai1^tg/tg cultures showing a significant increase in immature myeloid cells in the Snai1^tg/tg cultures at the first round of culture. (G-H) A similar increase in immature myeloid cells was also observed after the second round of replating. (A,C,D,F,H) Data are represented as mean + standard error of the mean; n = 3 biological replicates. *P < .05, **P < .01, ***P < .001 Student 2-tailed unpaired t test.